Molecular searches use one of several forms of complementarity to identify the macromolecules of interest among a large number of other molecules. Complementarity is the sequence-specific or shaped-specific molecular recognition that occurs when two molecules bind together. Complementarity between a probe molecule and a target molecule can result in the formation of a probe-target complex. This complex can then be located if the probe molecules are tagged with a detectible entity such as a chromophore, fluorophore, radioactivity, or an enzyme. There are several types of hybrid molecular complexes that can exist. A single-stranded DNA (ssDNA) probe molecule can form a double-stranded, base pair hybrid with an ssDNA target if the probe sequence is the reverse complement of the target sequence. An ssDNA probe molecule can form a double-stranded, base-paired hybrid with an RNA target if the probe sequence is the reverse complement of the target sequence. An antibody probe molecule can form a complex with a target protein molecule if the antibody's antigen-binding site can bind to an epitope on the target protein. There are two important features of hybridization reactions. First, the hybridization reactions are specific in that the probes will only bind to targets with a complementary sequence, or in the case of proteins, sites with the correct three-dimensional shape. Second, hybridization reactions will occur in the presence of large quantities of molecules similar but not identical to the target. A probe can find one molecule of a target in a mixture of a zillion of related but non-complementary molecules.
There are many research and commercially available protocols and devices that use hybridization reactions and employ some similar experimental steps. For example microarray (or DNA chip) based hybridization uses various probes which enable the simultaneous analysis of thousands of sequences of DNA for genetic and genomic research and for diagnosis. Most DNA microarray fabrications employ a similar experimental approach. The probe DNA with a defined identity is immobilized onto a solid medium. The probe is then allowed to hybridize with a mixture of nucleic acid sequences, or conjugates, that contain a detectable label. The signal is then detected and analyzed. Variations of this approach are available for RNA-DNA and protein-protein hybridizations and those hybridization techniques involving tissue sections that are immobilized on a support. In all of these protocols, the hybridization solution is placed directly on the support that contains the immobilized DNA or tissue section.
The hybridization reaction is usually performed in a warm environment and there are several ways to prevent evaporation and inadvertent contamination of the hybridization solution that is on the support. Cover slips have been placed directly on the solution, but the weight of the cover slip displaces the solution and minimizes the amount of solution that is in contact with the immobilized component. Devices are commercially available that form a chamber around the support to allow a desired volume of hybridization solution to be placed on the support. The support is then completely covered. With these devices, there is a problem of hybridization non-uniformity due to formation of concentration gradients resulting in unevenly dispersed conjugates. Thus, there is a desire to form a chamber that provides even dispersal throughout the hybridization solution during the reaction process.
Microfluidic devices are now being used to conduct biomedical research and create clinically useful technologies having a number of significant advantages. First, because the volume of fluids within these channels is very small, usually several nanoliters, the amount of reagents and analytes used is quite small. This is especially significant for expensive reagents. The fabrications techniques used to construct microfluidic devices are relatively inexpensive and are very amenable both to highly elaborate, multiplexed devices and also to mass production. In a manner similar to that for microelectronics, microfluidic technologies enable the fabrication of highly integrated devices for performing several different functions on the same substrate. Common fluids used in microfluidic devices include whole blood samples, bacterial cell suspensions, protein or antibody solutions and various buffers. Microfluidic devices can be used to obtain a variety of interesting measurements including molecular diffusion coefficients, fluid viscosity, pH, chemical binding coefficients, and enzyme reaction kinetics. Other applications for microfluidic devices include capillary electrophoresis, isoelectric focusing, immunoassays, flow cytometry, sample injection of proteins for analysis via mass spectrometry, PCR amplification, DNA analysis, cell manipulation, cell patterning, and chemical gradient formation.